Human adult spermatogonial stem cells (hSSCs) must balance self-renewal and differentiation. To understand how this is achieved, we profiled DNA methylation and open chromatin (ATAC-seq) in SSEA4⁺ hSSCs, analyzed bulk and single-cell RNA transcriptomes (RNA-seq) in SSEA4⁺ hSSCs and differentiating c-KIT⁺ spermatogonia, and performed validation studies via immunofluorescence. First, DNA hypomethylation at embryonic developmental genes supports their epigenetic "poising" in hSSCs for future/embryonic expression, while core pluripotency genes (OCT4 and NANOG) were transcriptionally and epigenetically repressed. Interestingly, open chromatin in hSSCs was strikingly enriched in binding sites for pioneer factors (NFYA/B, DMRT1, and hormone receptors). Remarkably, single-cell RNA-seq clustering analysis identified four cellular/developmental states during hSSC differentiation, involving major transitions in cell-cycle and transcriptional regulators, splicing and signaling factors, and glucose/mitochondria regulators. Overall, our results outline the dynamic chromatin/transcription landscape operating in hSSCs and identify crucial molecular pathways that accompany the transition from quiescence to proliferation and differentiation.

中文翻译：

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染色质和单细胞RNA序列分析揭示了人类精原干细胞发育过程中的动态信号和代谢转变。

人类成年精原干细胞（hSSC）必须平衡自我更新和分化。为了理解如何实现这一点，我们异型DNA甲基化和染色质开放（ATAC-SEQ）在SSEA4 ⁺ hSSCs，分析散装和SSEA4单细胞RNA转录（RNA-SEQ）⁺ hSSCs和分化的c-kit ⁺精原细胞，并通过免疫荧光进行验证研究。首先，胚胎发育基因的DNA低甲基化支持hSSC中它们的表观遗传“平衡”，以用于将来/胚胎表达，而核心多能性基因（OCT4和NANOG）在转录和表观遗传上受到抑制。有趣的是，hSSC中的开放染色质显着富集了先驱因子（NFYA / B，DMRT1和激素受体）的结合位点。值得注意的是，单细胞RNA-seq聚类分析确定了hSSC分化过程中的四个细胞/发育状态，涉及细胞周期和转录调节因子，剪接和信号传导因子以及葡萄糖/线粒体调节因子的主要转变。全面的，